Recent discoveries of genes involved in prevalent genetic-based human diseases have stimulated interest in understanding the molecular and biochemical basis for these disease processes. For example, a number of neurological syndromes and encephalomyopathies have been attributed to mutations in genes encoding mitochondrial electron transfer components. These results have stifled interest in understanding the biogenesis and function of individual mitochondrial components. As with most complex systems, the molecular processes involved in the biogenesis and function of these electron transfer components are likely to be elucidated by studying analogous simplified systems that are more amenable to genetic and biochemical analyses. Such a simplified system is described, a bacterium that is closely related to the ancestral mitochondrial endosymbiont; seven genes involved specifically in the biogenesis of c- type cytochromes have already been isolated (called helABCDX and ccl1 and ccl2). The c-type cytochromes possess heme that is covalently ligated to the apocytochrome and these cytochromes are present in distinct compartments within eucaryotes (i.e., mitochondrial intermembrane space) and procaryotes (i.e., external to the cytoplasmic membrane). Major questions that remain concern what factors are needed for their assembly and how these individual factors are themselves transported to the proper compartments. Specifically, using appropriate mutants and/or wild type strains we will: (1) Develop and use heme reporters to further study a putative heme transporter encoded by helABC genes (and the defects in Ccl- strains). (2) Isolate heme-correcting delta-helAB mutants (to test the heme transporter function for helABC). (3) Produce and use antisera to the Hel and Ccl proteins to investigate localization and macromolecular complex interactions. (4) Overproduction, purification and functional analyses of HelX, a periplasmic thioredoxin-like protein. The long-term objective of this work is to understand at a detailed molecular level the specific proteins and events that are required for the biogenesis of c-type cytochromes. Moreover, since heme is required for many different processes in eucaryotes and procaryotes, the mechanism by which heme is transported across bilayer membranes from its site of biosynthesis, the mitochondrial matrix or the bacterial cytoplasm, is a fundamental question in biology. Completion of the specific aims proposed here will answer questions concerning intracellular heme targeting, specific red-ox reactions within the cell, and cytochrome c biogenesis. A combination of bacterial genetics, immunological analyses, and molecular biology will be used to accomplish these goals.